Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2.

RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter-defensive measures by producing virulence determinants called viral-suppressors-of-RNAi (VSRs). Interestingly, in spite of dominance of interferon-based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double-stranded RNA genome. Intriguingly, a time point-dependent bimodal regulation of RNAi was observed in RV-infected cells, where short interfering RNA (siRNA)-based RNAi was rendered non-functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA-mediated RNAi, via ubiquitin-proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.

Rotavirus-induced miR-142-5p elicits proviral milieu by targeting non-canonical transforming growth factor beta signalling and apoptosis in cells.

MicroRNA (miRNA) expression is significantly influenced by viral infection, because of either host antiviral defences or proviral factors resulting in the modulation of viral propagation. This study was undertaken to identify and analyse the significance of cellular miRNAs during rotavirus (SA11 or KU) infection. Sixteen differentially regulated miRNAs were identified during rotavirus infection of which hsa-miR-142-5p was up-regulated and validated by quantitative polymerase chain reaction. Exogenous expression of miR-142-5p inhibitor resulted in a significant reduction of viral titer indicating proviral role of miR-142-5p. Functional studies of hsa-miR-142-5p identified its role in transforming growth factor beta (TGFß) signalling as TGFß receptor 2 and SMAD3 were degraded during both hsa-miR-142-5p overexpression and rotavirus infection. TGFß is induced during rotavirus infection, which may promote apoptosis by activation of non-canonical pathways in HT29 cells. However, up-regulated miR-142-5p resulted in the inhibition of TGFß-induced apoptosis suggesting its anti-apoptotic function. Rotavirus NSP5 was identified as a regulator of miR-142-5p expression. Concurrently, NSP5-HT29 cells showed inhibition of TGFß-induced apoptosis and epithelial to mesenchymal transition by blocking non-canonical pathways. Overall, the study identified proviral function of hsa-miR-142-5p during rotavirus infection. In addition, modulation of TGFß-induced non-canonical signalling in microsatellite stable colon cancer cells can be exploited for cancer therapeutics.

Rotaviral enterotoxin nonstructural protein 4 targets mitochondria for activation of apoptosis during infection.

Viruses have evolved to encode multifunctional proteins to control the intricate cellular signaling pathways by using very few viral proteins. Rotavirus is known to express six nonstructural and six structural proteins. Among them, NSP4 is the enterotoxin, known to disrupt cellular Ca(2+) homeostasis by translocating to endoplasmic reticulum. In this study, we have observed translocation of NSP4 to mitochondria resulting in dissipation of mitochondrial membrane potential during virus infection and NSP4 overexpression. Furthermore, transfection of the N- and C-terminal truncated NSP4 mutants followed by analyzing NSP4 localization by immunofluorescence microscopy identified the 61-83-amino acid region as the shortest mitochondrial targeting signal. NSP4 exerts its proapoptotic effect by interacting with mitochondrial proteins adenine nucleotide translocator and voltage-dependent anion channel, resulting in dissipation of mitochondrial potential, release of cytochrome c from mitochondria, and caspase activation. During early infection, apoptosis activation by NSP4 was inhibited by the activation of cellular survival pathways (PI3K/AKT), because PI3K inhibitor results in early induction of apoptosis. However, in the presence of both PI3K inhibitor and NSP4 siRNA, apoptosis was delayed suggesting that the early apoptotic signal is initiated by NSP4 expression. This proapoptotic function of NSP4 is balanced by another virus-encoded protein, NSP1, which is implicated in PI3K/AKT activation because overexpression of both NSP4 and NSP1 in cells resulted in reduced apoptosis compared with only NSP4-expressing cells. Overall, this study reports on the mechanism by which enterotoxin NSP4 exerts cytotoxicity and the mechanism by which virus counteracts it at the early stage for efficient infection.

Spectrum of respiratory viruses circulating in eastern India: prospective surveillance among patients with influenza-like illness during 2010-2011.

In developing countries, viruses causing respiratory disease are a major concern of public health. During January 2010-December 2011, 2,737 patients with acute respiratory infection from the outpatient departments as well as patients admitted to hospitals were screened for different respiratory viruses. Nasal and or throat swabs were collected and transported to the laboratory where initial screening of influenza A and influenza B viruses was performed. The samples were tested further for influenza C virus, parainfluenza viruses 1-4, human rhinovirus, metapneumovirus and respiratory syncytial virus by conventional RT- PCR. The study revealed that the majority of the patients were under 5 years of age; both due to their higher susceptibility to respiratory infections and presentation to hospitals. Out of 2,737 patients enrolled in this study, 59% were found positive for one or more respiratory viruses. Influenza B infection was detected in 12% of patients followed by influenza A (11.7%), respiratory syncytial virus (7.1%), parainfluenza virus-2 (6%), metapneumovirus (3%), parainfluenza virus-3 (1%), parainfluenza virus-4 (0.6%), parainfluenza virus-1 (0.3%), influenza C (0.2%) and human rhinovirus (0.2%). Distinct seasonal infection was observed only for influenza A and influenza B viruses.

Surveillance in Eastern India (2007-2009) revealed reassortment event involving NS and PB1-F2 gene segments among co-circulating influenza A subtypes.

BACKGROUND: Influenza A virus encodes for eleven proteins, of which HA, NA, NS1 and PB1-F2 have been implicated in viral pathogenicity and virulence. Thus, in addition to the HA and NA gene segments, monitoring diversity of NS1 and PB1-F2 is also important. METHODS: 55 out of 166 circulating influenza A strains (31 H1N1 and 24 H3N2) were randomly picked during 2007-2009 and NS and PB1-F2 genes were sequenced. Phylogenetic analysis was carried out with reference to the prototype strains, concurrent vaccine strains and other reference strains isolated world wide. RESULTS: Comparative analysis of both nucleotide and deduced amino acid sequences, revealed presence of NS gene with A/PR/8/34(H1N1)-like mutations (H4N, Q21R, A22V, K44R, N53D, C59R, V60A, F103S and M106I) in both RNA-binding and effector domain of NS1 protein, and G63E, the HPAI-H5N1-like mutation in NEP/NS2 of five A/H1N1 strains of 2007 and 2009. NS1 of other A/H1N1 strains clustered with concurrent A/H1N1 vaccine strains. Of 31 A/H1N1 strains, five had PB1-F2 similar to the H3N2 strains; six had non-functional PB1-F2 protein (11 amino acids) similar to the 2009 pandemic H1N1 strains and rest 20 strains had 57 amino acids PB1-F2 protein, similar to concurrent A/H1N1 vaccine strain. Interestingly, three A/H1N1 strains with H3N2-like PB1-F2 protein carried primitive PR8-like NS gene. Full gene sequencing of PB1 gene confirmed presence of H3N2-like PB1 gene in these A/H1N1 strains. CONCLUSION: Overall the study highlights reassortment event involving gene segments other than HA and NA in the co-circulating A/H1N1 and A/H3N2 strains and their importance in complexity of influenza virus genetics. In contrast, NS and PB1-F2 genes of all A/H3N2 eastern India strains were highly conserved and homologous to the concurrent A/H3N2 vaccine strains suggesting that these gene segments of H3N2 viruses are evolutionarily more stable compared to H1N1 viruses.

Synchronized Orchestration of miR-99b and let-7g Positively Regulates Rotavirus Infection by Modulating Autophagy.

Rotavirus (RV), the major etiological agent of viral gastroenteritis in young children, kills over 200 thousand infants each year. In spite of available vaccines, rotaviral diarrhoea is still a major problem in developing countries of Asia and Africa. Therefore, the studies on RV infection and host antiviral responses are warranted. The active correlation between virus infection and activation of autophagy machinery and positive influence of autophagy on RV replication have been documented recently. Previous study from our group showed dysregulation of several cellular miRNAs during RV infection, though their significance remained largely unknown. Since cellular microRNAs (miRNAs) have been implicated in the control of several fundamental biological processes including stress response and autophagy, we focused on two miRNAs, miR-99b and let-7g, and analyzed their function to gain insight into the miRNA-autophagy crosstalk during RV infection. This study shows that RV suppresses let-7g expression but enhances miR-99b that in turn augment major autophagy regulators. Ectopic expression of let-7g and knockdown of miR-99b resulted in inhibition of autophagy, hence, reduction of RV replication. Overall, our study highlights new mechanistic insights for understanding the role of miRNAs in modulating RV infection and possibility of using RNA interference as an antiviral therapeutic target.

Rotavirus infection induces G1 to S phase transition in MA104 cells via Ca⁺²/Calmodulin pathway.

Viruses, obligate cellular parasites rely on host cellular functions and target the host cell cycle for their own benefit. In this study, effect of rotavirus infection on cell cycle machinery was explored. We found that rotavirus (RV) infection in MA104 cells induces the expression of cyclins and cyclin dependent kinases and down-regulates expression of CDK inhibitors, resulting in G1 to S phase transition. The rotavirus induced S phase accumulation was found to be concurrent with induction in expression of calmodulin and activation of CaMKI which is reported as inducer of G1-S phase transition. This cell cycle manipulation was found to be Ca(+2)/Calmodulin pathway dependent. The physiological relevance of G1 to S phase transition was established when viral gene expressions as well as viral titers were found to be increased in S phase synchronized cells and decreased in G0/G1 phase synchronized cells compared to unsynchronized cells during rotavirus infection.

MAVS protein is attenuated by rotavirus nonstructural protein 1.

Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.